In primary cultures of bovine chromaffin cells, commercially available preparation of Alpha-bungarotoxin inhibit the acetylcholine (ACh)- or nicotine-evoked release of endogenous catecholamines. The potency of different lots of Alpha-bungarotoxin in not related to the Alpha-bungarotoxin peptide content but to that of another peptide (termed P-4 bungarotoxin) present as an impurity in the Alpha-bungarotoxin preparations. P-4 Bungarotoxin was isolated and purified to homogeneity by high-pressure liquid chromatography (HPLC). Homogeneity was established by a variety of mean, including polyacrylamide gel electrophoresis, HPLC, end carboxy group analysis and NH2-terminal amino acid sequence. Purified P-4 bungarotoxin contains approximately 121 amino acid residues, and it is different in its amino composition, molecular weight, and amino acid sequence from Alpha-bungarotoxin and Beta-bungarotoxin. P-4 Bungarotoxin (IC50 of about 1 nM) blocked the ACh-induced release of endogenous catecholamines but failed to block the KCl-induced catecholamine release. Although P-4 bungarotoxin is endowed with phospholipase A2 activity, its effect on ACh-evoked catecholamine release persists when the phospholipase activity is blocked (99.9%) by treatment of the toxin with p-bromophenacyl bromide. P-4 Bungarotoxin may represent a useful tool with which to study nicotinic receptor function in sympathetic and central nervous system neurons.